Cryptococcus neoformans is a pathogenic, encapsulated yeast of worldwide distribution with a marked predilection for the central nervous system. Cryptococcal meningitis is uniformly fatal if untreated and, even with adequate treatment, causes high morbitidy and mortality. Although some virulence factors of C. neoformans have been described, knowledge of the manner in which it causes disease is incomplete. This proposal aims to detect potential virulence mechanisms by screening a selection of isolates for enzymatic activities related to virulence in other systems. Causal association between the enzyme and virulence will be tested by examining the effect of the trait on virulence in isogenic strains of C. neoformans. Genetic analysis and biochemical characterization of identified virulence factors will be performed. Molecular cloning of C. neoformans DNA, especially fragments coding for virulence associated traits, will be carried out in E. coli and in Saccharomyces. Finally, a transformation system in Cryptococcus, perhaps using a plasmid carrying an autologous mitochondrial ars and G 418 resistance, will be established so that contributions to virulence by individual genes or gene products can be assayed in vivo. Identification of virulence mechanisms introduces the possibility of ablating those functions as an approach to therapy.